Multiple inhibitory receptors may play a role in the weak or absent CD8+ T-cell response in chronic hepatitis B virus (HBV) infection. Yet few receptors have been characterized in detail and little is known about their complex regulation. In the present study, we investigated the role of the signaling lymphocyte activation molecule (SLAM)-related receptor CD244 and of programmed death 1 (PD-1) in HBV infection in 15 acutely and 66 chronically infected patients as well as 9 resolvers and 21 healthy controls. The expression of CD244, PD-1, and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) was analyzed in virus-specific CD8+ T-cells derived from peripheral blood or liver using major histocompatibility complex class I pentamers targeting immunodominant epitopes of HBV, Epstein-Barr-virus (EBV), or influenza virus (Flu). In chronic HBV infection, virus-specific CD8+ T-cells expressed higher levels of CD244 both in the peripheral blood and liver in comparison to the acute phase of infection or following resolution. CD244 was expressed at similarly high levels in EBV infection, but was low on Flu-specific CD8+ T-cells. In chronic HBV infection, high-level CD244 expression coincided with an increased expression of PD-1. The inhibition of the CD244 signaling pathway by antibodies directed against either CD244 or its ligand CD48 resulted in an increased virus-specific proliferation and cytotoxicity as measured by the expression of CD107a, interferon-γ, and tumor necrosis factor-α in CD8+ T-cells.

CONCLUSIONS

CD244 and PD-1 are highly coexpressed on virus-specific CD8+ T-cells in chronic HBV infection and blocking CD244 or its ligand CD48 may restore T-cell function independent of the PD-1 pathway. CD244 may thus be another potential target for immunotherapy in chronic viral infections.

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.

Several receptors and counter-receptor pairs on T cells and on antigen-presenting cells (APCs) deliver costimulatory signals to T cells during antigen presentation. The CD28 receptor on T cells with its ligand B7 represents one of the best characterized and most important examples of this costimulation. We show here that interleukin 12 (IL-12), a cytokine also produced by APCs (monocyte/macrophages and B cells) and active on T and natural killer cells, has a strong synergistic effect with the B7/CD28 interaction in inducing proliferation and cytokine production in both mitogen-activated and freshly isolated peripheral blood T cells. Together with anti-CD28 antibodies, IL-12 induces proliferation of T cells to levels higher than those obtained with IL-2 stimulation and it is effective at IL-12 concentrations 100- to 1,000-fold lower than effective concentrations of IL-2. The proliferative effect of anti-CD28 and IL-12 is resistant to moderate doses of cyclosporin A and is largely independent of endogenous IL-2, IL-12, in synergy with anti-CD28 or B7-transfected cells, is most effective in inducing interferon gamma (IFN-gamma) production, but production of tumor necrosis factor alpha and granulocyte/macrophage colony-stimulating factor is also observed. IL-12-induced IFN-gamma production in peripheral blood mononuclear cells is inhibited by the chimeric molecule CTLA-4 immunoglobulin, which prevents binding of CD28 to B7, suggesting that endogenous B7 on the mononuclear cells and IL-12 cooperate in inducing IFN-gamma production. IL-10 inhibits both IL-12 production and B7 expression on monocytes. These two effects are largely responsible for the ability of IL-10, acting on accessory cells, to inhibit IFN-gamma production by lymphocytes, because anti-CD28 antibodies and IL-12 can reverse the inhibitory effect of IL-10 on IFN-gamma production. Our results in vitro suggest that the synergy between B7 and IL-12, a surface antigen and a soluble product of APCs, respectively, plays a role in regulating T cell activation and immune response in the microenvironment of inflamed tissues.

The cytokine interleukin (IL) 12 stimulates T cell and natural killer cell production of interferon (IFN) gamma and inhibits T cell production of IL-4. We investigated the effects of IL-12 on cytokine gene expression, immunoglobulin (Ig)E, mucosal mast cell, and eosinophil responses, and the course of infection in mice inoculated with the nematode parasite Nippostrongylus brasiliensis, as well as the IFN-gamma dependence of these effects. IL-12 stimulated IFN-gamma and IL-10 gene expression during primary and secondary N. brasiliensis infections and inhibited IL-3, IL-4, IL-5, and IL-9 gene expression during primary infections but had little inhibitory effect during secondary infections. IL-12 inhibited IgE, mucosal mast cell, and blood and tissue eosinophil responses during primary infections, but only eosinophil responses during secondary infections. IL-12 enhanced adult worm survival and egg production during primary, but not secondary infections. IL-12 needed to be administered by day 4 of a primary infection to inhibit IgE and mucosal mast cell responses, and by day 6 to strongly inhibit eosinophil responses and to enhance worm survival and fecundity. Anti-IFN-gamma mAb inhibited the effects of IL-12 on IgE secretion, intestinal mucosal mastocytosis, and parasite survival and fecundity, but did not affect IL-12 inhibition of eosinophilia. These observations indicate that IL-12, if administered during the initiation of eosinophilia. These observations indicate that IL-12, if administered during the initiation of an immune response, can change the response from one that is characterized by the production of T helper (Th)2-associated cytokines to one characterized by the production of Th-1 associated cytokines. However, IL-12 treatment has less of an effect once the production of Th2-associated cytokines has become established. In addition, our results provide evidence that Th2-associated responses protect against, and/or Th1-associated responses exacerbate, nematode infections.

Two chemically mutagenized agerminative variants of Candida albicans were used to immunize mice against challenge with highly virulent cells of the parent strain. Although both mutants (Vir- 3 and Vir- 13) resulted in nonlethal infection and could be recovered from mouse organs for many days after the intravenous inoculation of 10(7) to 10(6) cells, significant protection to systemic challenge with virulent C. albicans was induced by only one (Vir- 3) of the two variants. Anticandidal resistance in Vir- 3-infected mice was associated with the occurrence in vivo of strong delayed-type hypersensitivity to Candida antigen, detection in vitro of highly fungicidal effector macrophages, and presence in the serum of a large proportion of Candida-reactive antibodies of the immunoglobulin G2a isotype. Bulk cultures of purified CD4+ lymphocytes from mice infected with either mutant were compared for their ability to produce gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-6 in vitro. After stimulation with specific antigen, CD4+ cells from Vir- 3-immunized mice released large amounts of the Th1-specific cytokines, IFN-gamma and IL-2, at a time when CD4+ cells from Vir- 13-infected mice predominantly secreted the characteristic Th2 cytokines, IL-4 and IL-6. These results were confirmed by quantitative analysis of cytokine-producing Th1 and Th2 cells. In addition, only mice infected with Vir- 3 displayed a high frequency of CD8+ cells with the potential for in vitro lysis of yeast-primed bone marrow macrophages. Purified CD4+ cells from Vir- 3-infected mice, but not a mixture of these cells with CD4+ lymphocytes from mice infected with Vir- 13, could adoptively transfer delayed-type hypersensitivity reactivity onto naive mice. Taken together, these data suggest that both Th1 and Th2 CD4+ lymphocytes may be activated during experimental C. albicans infection in mice.

Extent of O-2-release and chemiluminescence, attributed to singlet oxygen, has been compared in human monocytes and neutrophils during phagocytosis, stimulation by the surface-active agent phorbol myristate acetate, or contact with aggregated IgG in a model of immune complex disease. Monocytes generated O-2-and chemiluminescence with each of the three stimuli, although values were significantly less than those of neutrophils from the same individuals. Lymphocytes had no significant activity in either assay with any stimulus. Oxygen metabolites released from mononuclear phagocytes are highly reactive and could play a part in both the beneficial and detrimental aspects of chronic inflammation.

Protein antigens I, I/II, II, and III were prepared from Streptococcus mutans (serotype c). Their immunogenicities and protective effects against dental caries were investigated in 40 rhesus monkeys kept entirely on a human-type diet, containing about 15% sucrose. Antigens I, I/II and, to a lesser extent, antigen II induced significant reductions in dental caries, as compared with sham-immunized monkeys. This was achieved with 1 or 2 doses of antigen, the first of which was administered with adjuvant (Freund incomplete adjuvant or aluminum hydroxide). There was no reduction in caries in monkeys immunized with antigen III. The reduction in caries in the animals immunized with antigens I or I/II was comparable to that in monkeys immunized with whole cells. Protection against caries was associated predominantly with serum and gingival crevicular fluid immunoglobulin G antibodies, which appeared to be directed against the antigen I determinant, but antibodies to antigen II, though not to antigen III, were also protective.

BACKGROUND

Helicobacter pylori has been implicated as a possible etiologic factor in gastric cancer. This case control study was performed to determine the association between H. pylori and gastric cancer, taking into account the possibility of confounding by other background factors.

METHODS

Sera were collected from 112 incident case patients with gastric cancer and 103 control patients with nongastroenterological diseases, who were frequency-matched with respect to age and sex. Immunoglobulin G antibodies to H. pylori were identified using the HM-CAP immunoassay (Enteric Products Inc., Wesbury, NY).

RESULTS

The prevalence of H. pylori seropositivity was significantly higher (P = 0.002) among case patients than control patients. The odds ratio (OR) was 2.60 (95% confidence interval, 1.35-5.02). The increased OR associated with H. pylori infection was confined to tumors with a noncardia location (OR, 3.06) and men (OR, 4.27). OR increased with decreasing age at cancer diagnosis to reach 9.33 in patients < 60 years of age. Multivariate logistic regression analysis was used as control for potential confounding, but the elevated OR associated with H. pylori infection remained significantly increased.

CONCLUSIONS

The results support the hypothesis of H. pylori infection as an independent risk indicator of gastric cancer.

The platelet plays a pivotal role in maintaining vascular integrity. In a manner similar to leukocytes, platelets interact with selectins expressed on activated endothelium. P-selectin glycoprotein ligand 1 (PSGL-1) is the main P-selectin ligand expressed on leukocytes. Searching for platelet ligand(s), we used a P-selectin-immunoglobulin G (IgG) chimera to affinity purify surface-biotinylated proteins from platelet lysates. P-selectin-bound ligands were eluted with ethylenediaminetetraacetic acid. An approximately 210-kD biotinylated protein was isolated from both human neutrophil and platelet preparations. A band of the same size was also immunopurified from human platelets using a monoclonal anti-human PSGL-1 antibody and could be blotted with P-selectin-IgG. Under reducing conditions, both the predicted PSGL-1 approximately 210-kD dimer and the approximately 120-kD monomer were isolated from platelets. Comparative immunoelectron microscopy and Western blotting experiments suggested that platelet PSGL-1 expression is 25-100-fold lower than that of leukocytes. However, patients with chronic idiopathic thrombocytopenic purpura who harbor predominantly young platelets displayed greater expression, indicating that PSGL-1 expression may be decreased during platelet aging. By flow cytometry, thrombin-activated platelets from normal individuals exhibited greater expression than those unstimulated. An inhibitory anti-PSGL-1 antibody significantly reduced platelet rolling in mesenteric venules, as observed by intravital microscopy. Our results indicate that functional PSGL-1 is expressed on platelets, and suggest an additional mechanism by which selectins and their ligands participate in inflammatory and/or hemostatic responses.

The term 'neuromyelitis optica' ('Devic's syndrome', NMO) refers to a syndrome characterized by optic neuritis and myelitis. In recent years, the condition has raised enormous interest among scientists and clinical neurologists, fuelled by the detection of a specific serum immunoglobulin (Ig)G reactivity (NMO-IgG) in up to 80% of patients with NMO. These autoantibodies were later shown to target aquaporin-4 (AQP4), the most abundant water channel in the central nervous system (CNS). Here we give an up-to-date overview of the clinical and paraclinical features, immunopathogenesis and treatment of NMO. We discuss the widening clinical spectrum of AQP4-related autoimmunity, the role of magnetic resonance imaging (MRI) and new diagnostic means such as optical coherence tomography in the diagnosis of NMO, the role of NMO-IgG, T cells and granulocytes in the pathophysiology of NMO, and outline prospects for new and emerging therapies for this rare, but often devastating condition.

We have determined the complete nucleotide and deduced amino acid sequence of the major protein core of the human heparan sulfate proteoglycan HSPGGly-Asp sequences that could act as attachment sites for heparan sulfate glycosaminoglycans. Domain II is highly homologous to the LDL receptor and contains four repeats with perfect conservation of all 6 consecutive cysteines. Next is domain III which shares homology to the short arm of laminin A chain and contains four cysteine-rich regions intercalated among three globular domains. Domain IV, the largest module with greater than 2000 residues, contains 21 repeats of the immunoglobulin type as found in neural cell adhesion molecule. Near the beginning of this domain, there is a stretch of 29 hydrophobic amino acids which could allow the molecule to interact with the plasma membrane. Domain V, similar to the carboxyl-terminal globular G-domain of laminin A and to the related protein merosin, contains three globular regions and four EGF-like repeats. In situ hybridization and immunoenzymatic studies show a close association of this gene product with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Our studies show that this proteoglycan is present in all vascularized tissues and suggest that this unique molecule has evolved from the utilization of modular structures with adhesive and growth regulatory properties.

OBJECTIVE

To compare antibody responses to 23-valent pneumococcal vaccine (Pneumovax) in controls and patients with established rheumatoid arthritis (RA) treated with TNF blockers, methotrexate (MTX) or a combination of both.

METHODS

Patients with RA (n = 149) and healthy controls (n = 47) were vaccinated. Treatment with TNF blockers (etanercept or infliximab) and MTX was given to 50 patients, and 62 patients were treated with TNF blockers alone or with other DMARDs. MTX alone was given to 37 patients. Concentrations of immunoglobulin G (IgG) antibodies against pneumococcal capsular polysaccharides 23F and 6B were measured by enzyme-linked immunoassay before and 4-6 weeks after vaccination. An immune response was defined as a twofold or higher increase in antibody concentration following vaccination.

RESULTS

Prevaccination antibody levels for both 23F and 6B were similar in the patient groups. Antibody concentrations after vaccination increased significantly in all groups. Patients treated with TNF blockers without MTX showed better immune responses than those treated with TNF blockers in combination with MTX (P = 0.037 for 23F and P = 0.004 for 6B) or MTX alone (P<0.001 for both 23F and 6B). RA patients given MTX alone had the lowest immune responses. Prednisolone treatment did not influence the responses.

CONCLUSIONS

Patients treated with TNF blockers and controls showed similar responses to vaccination. In contrast, patients treated with MTX had reduced responses regardless of anti-TNF treatment. The findings do not argue against the use of pneumococcal vaccination in RA patients undergoing treatment with TNF blockers.

BACKGROUND

In vitro, mesenchymal stem cells (MSCs) have demonstrated a low immunogenic profile. In this study, we tested the immune response to allogeneic MSCs in immunocompetent swines both in vitro and in vivo.

METHODS

Major histocompatibility complex-controlled swine leukocyte antigen (SLA) and SLA were used as donor and recipient, respectively. In vitro, proliferative responses were tested by mixed lymphocyte reaction (MLR) or cocultures and cytokine profiling by enzyme-linked immunosorbent assay. In vivo, allogeneic MSCs were injected in cardiac infarcted area (n=3) and compared with subcutaneous injections of either MSCs (n=2) or peripheral blood mononuclear cells (PBMCs; n=2). Two additional animals received a skin graft as controls. No immunosuppression was used. Specific antidonor humoral responses were tested by flow cytometry and complement-dependent cytotoxicity assay.

RESULTS

In vitro, either unstimulated MSCs or interferon (IFN)-gamma stimulated MSC failed to elicit a proliferative response (stimulation index: 1.23 vs. 1.12 vs. 36.9 for controls, P<.001). Concomitantly to the absence of proliferation to MSCs, low production of IFN-gamma and interleukin-2 was evidenced in supernatants while the production of Th2 cytokines was comparable to controls. In vivo, all animals receiving skin grafts, subcutaneous PBMCs and intracardiac MSCs injections developed donor-specific cellular and humoral responses (immunoglobulins M and G) with antibody-complement-mediated cytotoxicity. Subcutaneous MSCs injection needed a rechallenge to similarly develop a cytotoxic humoral response.

CONCLUSIONS

Intracardiac allogeneic porcine mesenchymal stem cells elicit an immune response despite their low immunogenic profile in vitro. This result suggests that in vivo characteristics of allogeneic MSCs might differ and emphasizes the importance of pursuing research both in vitro and in vivo.

We evaluated a patient with disseminated Mycobacterium tuberculosis and Mycobacterium chelonae infection, of which he died. He also developed autoimmune (type I) diabetes and primary hypothyroidism. His serum contained a high titer of immunoglobulin G autoantibody to interferon-gamma (IFN-gamma) capable of blocking in vitro responses to this cytokine by peripheral blood mononuclear cells from normal donors. These results suggest that autoantibodies to IFN-gamma can induce susceptibility to disseminated mycobacterial infection, which may be refractory to chemotherapy.

BACKGROUND

Fabry disease is an X-linked inherited disorder that is caused by excessive lysosomal globotriaosylceramide (CTH) storage due to a deficiency in alpha-galactosidase A (alpha-Gal A). Two recombinant enzyme preparations have been approved as treatment modality. We studied emergence and properties of alpha-Gal A antibodies in treated patients.

METHODS

During the first 6 to 12 months of intravenous administration of recombinant enzymes (rh-alpha-Gal A) formation of antibodies was studied in 18 adult Fabry patients (two females).

RESULTS

The female patients did not develop detectable amounts of antibodies following enzyme therapy. After 6 months of treatment with either agalsidase alpha or beta, 11/16 male patients showed high titers of immunoglobulin G (IgG) antibodies that cross-react in vitro similarly with both recombinant enzymes. The anti-rh-alpha-Gal A IgG neutralizes rh-alpha-Gal A activity in vitro for 65% to 95%. During infusion with rh-alpha-Gal A, circulating enzyme-antibody complexes are formed and these complexes are taken up by leukocytes in the peripheral blood. After 6 months of treatment all IgG-negative patients showed a significant (P < 0.01) reduction of urinary CTH (1890 +/- 797 to 603 +/- 291 nmol CTH/24hr urine), compared to IgG-positive patients (mean increase from 2535 +/- 988 to 2723 +/- 1212), suggesting a negative effect of circulating antibodies on renal tubular CTH clearance.

CONCLUSIONS

Emergence of antibodies with in vivo neutralizing capacities is frequently encountered in treated Fabry disease patients. Complete cross-reactivity of these antibodies suggests that it is unlikely that switching from one to the other recombinant protein prevents the immune response and related effects. Further studies on the clinical implications of alpha-Gal A antibodies are essential.

T lymphocytes recognize cell-bound antigens in the molecular context of the self major histocompatibility complex (MHC) gene products through the surface T-cell receptor(s). The minimal component of the T-cell receptor is a heterodimer composed of alpha and beta subunits, each of relative molecular mass (Mr) approximately 45,000 (refs 1-3). Recently, complementary DNA clones encoding these subunits have been isolated and characterized along with that of a third subunit of unknown function, termed gamma (refs 4-9). These studies revealed a primary structure for each subunit that was clearly similar to that of immunoglobulin and indicated a somatic rearrangement of corresponding genes that are also immunoglobulin-like. Recently, the analysis of the sequence organization of the T-cell receptor beta-chain and T-cell-specific gamma-chain gene families has been reported. We now present an initial characterization of the murine T-cell receptor alpha-chain gene family, and conclude that although it is clearly related to the gene families encoding immunoglobulins, T-cell receptor beta-chains and also T-cell gamma-chains, it shows unique characteristics. There is only a single constant (C) region gene segment, which is an exceptionally large distance (approximately 20-40 kilobases (kb) in the cases studied here) from joining (J) gene segments. In addition, the J cluster and the variable (V) segment number seen to be very large. Finally, in the case studied here, a complete alpha-chain gene shows no somatic mutation and can be assembled directly from V alpha, J alpha and C alpha segments without inclusion of diversity (D alpha) segments.

BACKGROUND

Hepatitis E virus (HEV) is prevalent and causes disease worldwide, but its epidemiological profile is only partially understood.

METHODS

We used an enzyme immunoassay to measure anti-HEV immunoglobulin G antibodies in 18,695 serum samples collected in the Third National Health and Nutrition Examination Survey. We calculated estimates of HEV seroprevalence and examined associations with putative risk factors.

RESULTS

The seroprevalence of HEV in the civilian noninstitutionalized United States (US) population during the period from 1988 through 1994 was 21.0% (95% confidence interval [CI], 19.0%-22.9%). Among US-born individuals, males, non-Hispanic whites, and individuals residing in the Midwest and/or in metropolitan areas had the highest seroprevalence estimates. Having a pet in the home (odds ratio [OR], 1.19 [95% CI, 1.01-1.40]) and consuming liver or other organ meats more than once per month (OR, 1.38 [95% CI, 1.01-1.88]) were significantly associated with increased odds of HEV seropositivity.

CONCLUSIONS

Exposure to HEV is common in the US population, although hepatitis E is rarely reported. Having pets and consuming organ meats may play a role in HEV transmission in the United States, but other mechanisms of transmission may also exist. HEV may be considered a possible etiologic agent of acute and chronic hepatitis in US patients reporting no travel history.

There is a direct correlation between protein levels and disease states in human serum, which makes it an attractive target for sensors and diagnostics. However, this is challenging because serum features more than 20,000 proteins, with an overall protein content greater than 1 mM. Here we report a sensor based on a hybrid synthetic-biomolecule that uses arrays of green fluorescent protein and nanoparticles to detect proteins at biorelevant concentrations in both buffer and human serum. Distinct and reproducible fluorescence-response patterns were obtained from five serum proteins (human serum albumin, immunoglobulin G, transferrin, fibrinogen and a-antitrypsin), both in buffer and when spiked into human serum. Using linear discriminant analysis we identified these proteins with an identification accuracy of 100% in buffer and 97% in human serum. The arrays were also able to discriminate between different concentrations of the same protein, as well as a mixture of different proteins in human serum.

The clinical associations of glycine receptor antibodies have not yet been described fully. We identified prospectively 52 antibody-positive patients and collated their clinical features, investigations and immunotherapy responses. Serum glycine receptor antibody endpoint titres ranged from 1:20 to 1:60 000. In 11 paired samples, serum levels were higher than (n = 10) or equal to (n = 1) cerebrospinal fluid levels; there was intrathecal synthesis of glycine receptor antibodies in each of the six pairs available for detailed study. Four patients also had high glutamic acid decarboxylase antibodies (>1000 U/ml), and one had high voltage-gated potassium channel-complex antibody (2442 pM). Seven patients with very low titres (<1:50) and unknown or alternative diagnoses were excluded from further study. Three of the remaining 45 patients had newly-identified thymomas and one had a lymphoma. Thirty-three patients were classified as progressive encephalomyelitis with rigidity and myoclonus, and two as stiff person syndrome; five had a limbic encephalitis or epileptic encephalopathy, two had brainstem features mainly, two had demyelinating optic neuropathies and one had an unclear diagnosis. Four patients (9%) died during the acute disease, but most showed marked improvement with immunotherapies. At most recent follow-up, (2-7 years, median 3 years, since first antibody detection), the median modified Rankin scale scores (excluding the four deaths) decreased from 5 at maximal severity to 1 (P < 0.0001), but relapses have occurred in five patients and a proportion are on reducing steroids or other maintenance immunotherapies as well as symptomatic treatments. The glycine receptor antibodies activated complement on glycine receptor-transfected human embryonic kidney cells at room temperature, and caused internalization and lysosomal degradation of the glycine receptors at 37°C. Immunoglobulin G antibodies bound to rodent spinal cord and brainstem co-localizing with monoclonal antibodies to glycine receptor-α1. Ten glycine receptor antibody positive samples were also identified in a retrospective cohort of 56 patients with stiff person syndrome and related syndromes. Glycine receptor antibodies are strongly associated with spinal and brainstem disorders, and the majority of patients have progressive encephalomyelitis with rigidity and myoclonus. The antibodies demonstrate in vitro evidence of pathogenicity and the patients respond well to immunotherapies, contrasting with earlier studies of this syndrome, which indicated a poor prognosis. The presence of glycine receptor antibodies should help to identify a disease that responds to immunotherapies, but these treatments may need to be sustained, relapses can occur and maintenance immunosuppression may be required.

The gastrointestinal mucosa is the largest reservoir of macrophages in the body. These important effector cells are derived from blood monocytes that are recruited to the lamina propria by endogenous chemoattractants in the non-inflamed mucosa and by inflammatory chemokines and bacterial products during inflammation. In the non-inflamed mucosa, newly recruited pro-inflammatory monocytes are exposed to lamina propria stromal (extracellular matrix) factors that induce phenotypic and functional differentiation into non-inflammatory macrophages. As a consequence of this differentiation, resident lamina propria macrophages are strikingly downregulated for the expression of innate response receptors, such as the receptors for lipopolysaccharide, immunoglobulin G (IgG), and IgA, and the production of pro-inflammatory cytokines, including interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-alpha. Despite downregulated pro-inflammatory function, strong phagocytic and bactericidal activities remain intact. Thus, in the non-inflamed intestinal mucosa, lamina propria macrophages are non-inflammatory but retain avid scavenger and host defense functions, a unique but ideal phenotype and functional profile for effector cells in close proximity to immunostimulatory microorganisms and products.

Heavy and light polypeptide chains isolated from different specific antibodies to haptens and from (gamma)G-immunoglobulin of normal rabbits have been resolved into distinct, multiple components by disc electrophoresis in polyacrylamide gels in the presence of urea. In spite of the resolution of these chains into multiple bands, different specific antibodies and normal rabbit (gamma)G-immunoglobulin were indistinguishable from each other by this method.

Gastric Helicobacter mustelae was present in 100% of 11 adult female ferrets (Mustela putorius furo). The high immunoglobulin G antibody levels to H. mustelae in all ferrets showed a significant immune response to the organism. Urease mapping of the ferret stomach indicated that the bacteria heavily colonized the proximal duodenum and antrum and, to a lesser extent, the corpus. The histological gastritis observed coincided with presence of H. mustelae. Superficial gastritis was noted in the oxyntic gastric mucosa, whereas in the distal antrum the chronic inflammatory response occupied the full thickness of the mucosa. In the proximal antrum and transitional mucosa, focal glandular atrophy and regeneration were observed. Seven control specific-pathogen-free ferrets were not colonized with the bacteria, did not have detectable levels of immunoglobulin G H. mustelae antibody, and did not have H. mustelae-associated gastritis. The ferret lacks the polymorphonuclear-cell response seen in active chronic gastritis typically described with Helicobacter pylori gastritis in humans. However, the lesion in ferrets does closely resemble the diffuse antral gastritis seen in a subset of adults with H. pylori gastritis as well as children infected with H. pylori. Like H. pylori, H. mustelae adheres tightly to gastric mucosa. The ferret infected with H. mustelae, in addition to specific-pathogen-free uninfected control ferrets, will make longitudinal studies possible, enabling dissection of multiple host and environmental variables that influence the effect of H. mustelae colonization on progression and severity of gastroduodenal disease.

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine receptor γ chain. These mutations classically lead to complete absence of functional T and natural killer cell lineages as well as to intrinsically compromised B cell function. Although human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft-versus-host disease, and increased overall mortality. Here, 10 children were treated with autologous CD34(+) hematopoietic stem and progenitor cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow-up of 80 months (range, 54 to 107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral immunity only partially recovered but was sufficient in some patients to allow for withdrawal of immunoglobulin replacement; however, three patients developed antibiotic-responsive acute pulmonary infection after discontinuation of antibiotic prophylaxis and/or immunoglobulin replacement. One patient developed acute T cell acute lymphoblastic leukemia because of up-regulated expression of the proto-oncogene LMO-2 from insertional mutagenesis, but maintained a polyclonal T cell repertoire through chemotherapy and entered remission. Therefore, gene therapy for SCID-X1 without myelosuppressive conditioning effectively restored T cell immunity and was associated with high survival rates for up to 9 years. Further studies using vectors designed to limit mutagenesis and strategies to enhance B cell reconstitution are warranted to define the role of this treatment modality alongside conventional HSCT for SCID-X1.

OBJECTIVE

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to influence autoimmune diseases; however, its function in viral infection has not been well-defined. We therefore investigated the expression and regulatory function of Tim-3 in natural killer (NK) cells in chronic Hepatitis B (CHB) infection.

METHODS

Seventy-six CHB patients, 38 healthy controls, and 18 patients with fatty liver disease (FLD) were tested for Tim-3 expression on peripheral blood mononuclear cells (PBMCs) and in the liver tissue by flow cytometry and immunohistochemical stainning. The effects of HBV infection on Tim-3 expression in NK cells and the roles of Tim-3 in regulation of NK-cell function were also studied.

RESULTS

There was a significant increase of Tim-3 expression in PBMCs, circulating NK cells and liver infiltrating lymphocytes (LILs) from CHB patients compared to that of healthy controls and FLD patients. Increased Tim-3 expression was also detected in NK92 cells that had been transfected with a HBV expression vector and NK cells isolated from the liver of HBV transgenic mice. Importantly, blockage of Tim-3 signaling with anti-Tim-3 antibodies or Tim-3-Fc fusion proteins resulted in an increased cytotoxicity for NK92 cells compared to HepG2 and HepG2.2.15 cells, as well as an elevated interferon-gamma (IFN-gamma) production. Similarly, enhanced cytotoxicity was also observed in PBMCs or NK cells from CHB patients treated with the Tim-3 blockade ex vivo.

CONCLUSIONS

HBV infection can up-regulate Tim-3 expression in NK cells, which may in turn suppress NK-cell functions in CHB patients.